Insects and other pests cost farmers billions of dollars annually in crop losses and in the expense of keeping these pests under control. The losses caused by pests in agricultural production environments include decrease in crop yield, reduced crop quality, and increased harvesting costs.
Insects of the Order Coleoptera (coleopterans) are an important group of agricultural pests which cause extensive damage to crops each year. There are a number of beetles that cause significant economic damage; examples include Chrysomelid beetles (such as flea beetles and corn rootworms) and Curculionids (such as alfalfa weevils).
Flea beetles include a large number of genera (e.g., Altica, Apphthona, Argopistes, Disonycha, Epitrix, Longitarsus, Prodagricomela, Systena, Psylliodes, and Phyllotreta). Phyllotreta striolata includes the striped flea beetle. Phyllotreta cruciferae includes the canola flea beetle, the rape flea beetle, and the crucifer flea beetle. Canola, also known as rape, is an oil seed brassica (e.g., Brassica campestris, Brassica rapa, Brassica napus, and Brassica juncea).
Flea beetles include a large number of beetles that feed on the leaves of a number of grasses, cereals, and herbs. Phyllotreta cruciferae, Phyllotreta striolata, and Phyllotreta undulata, are particularly destructive annual pests that attack the leaves, stems, pods, and root tissues of susceptible plants. Psylliodes chrysocephala, a flea beetle, is also a destructive, biennial pest that attacks the stems and leaves of susceptible plants.
Chemical pesticides have provided effective pest control; however, the public has become concerned about contamination of food with residual chemicals and of the environment, including soil, surface water, and ground water. Working with pesticides may also pose hazards to the persons applying them. Stringent new restrictions on the use of pesticides and the elimination of some effective pesticides form the marketplace could limit economical and effective options for controlling costly pests.
In addition, the regular use of pesticides for the control of unwanted organisms can select for resistant strains. This has occurred in many species of economically important insects and other pests. The development of pesticide resistance necessitates a continuing search for new control agents having different modes of action.
Thus, there is an urgent need to identify new methods and compositions for controlling pests, such as the many different types of coleopterans that cause considerable damage to susceptible plants.
Certain strains of the soil microbe Bacillus thuringiensis (B.t.), a Gram-positive, spore-forming bacterium, can be characterized by parasporal crystalline protein inclusions. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and are specific in their toxic activity. These .delta.-endotoxins, which are produced by certain B.t. strains, are synthesized by sporulating cells. Certain types of B.t. toxins, upon being ingested by a susceptible insect, are transformed into biologically active moieties by the insect gut juice proteases. The primary target is cells of the insect gut epithelium, which are rapidly destroyed by the toxin.
Certain B.t. toxin genes have been isolated and sequenced. The cloning and expression of a B.t. crystal protein gene in Escherichia coli has been described in the published literature (Schnepf, H. E., H. R. Whiteley [1981] Proc. Natl. Acad. Sci. USA 78:2893-2897). U.S. Pat. No. 4,448,885 and U.S. Pat. No. 4,467,036 both disclose the expression of B.t. crystal protein in E. coli. In addition. with the use of genetic engineering techniques, new approaches for delivering these B.t. endotoxins to agricultural environments are under development, including the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. endotoxin delivery vehicles (Gaertner. F. H., L. Kim [1988] TIBTECH 6:S4-S7). Recombinant DNA-based B.t. products have been produced and approved for use. Thus, isolated B.t. endotoxin genes are becoming commercially valuable.
Until fairly recently, commercial use of B.t. pesticides has been largely restricted to a narrow range of lepidopteran(caterpillar)pests. Preparations of the spores and crystals of B. thuringiensis subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1 produces a crystalline .delta.-endotoxin which is toxic to the larvae of a number of lepidopteran insects. U.S. Pat. Nos. 4,990,332; 5,039,523; and 5,126,133 are among those which disclose B.t. toxins having activity against lepidopterans.
In recent years, however, new subspecies of B.t. have been identified, and investigators have discovered B.t. pesticides with specificities for a much broader range of pests. For example, other species of B.t., namely israelensis and morrisoni (a.k.a. tenebrionis, a.k.a. B.t. M-7, a.k.a. B.t. san diego), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, F. H. [1989] "Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms," in Controlled Delivery of Crop Protection Agents, R. M. Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255.). See also Couch, T. L. (1980) "Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis," Developments in Industrial Microbiology 22:61-76; and Beegle. C. C., (1978) "Use of Entomogenous Bacteria in Agroecosystems," Developments in Industrial Microbiology 20:97-104.
B.t. isolates having activity against dipterans are disclosed in, for example. U.S. Pat. Nos. 4,918,006; 5,275,815; and 5,298,245.
U.S. Pat. No. 5,151,363 and U.S. Pat. No. 4,948,734, for example, disclose B.t. isolates which have activity against nematodes.
Krieg, A., A. M. Huger, G. A. Langenbruch, W. Schnetter (1983) Z. ang. Ent. 96:500-508, describe a B.t. isolate named Bacillus thuringiensis var. tenebrionis, which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle, Leptinotarsa decemlineata, and Agelastica alni. U.S. Pat. Nos. 4,797,276 and 4,853,331 disclose B. thuringiensis strain tenebrionis which can be used to control coleopteran pests.
U.S. Pat. No. 4,764,372 discloses a novel B.t. isolate active against Coleoptera. It is known as B. thuringiensis M-7. U.S. Pat. No. 4,966,765 discloses the coleopteran-active Bacillus thuringiensis isolate B.t. PS86B1. U.S. Pat. No. 4,996,155 also discloses a novel B.t. isolate active against Coleoptera. This isolate is B.t. PS43F.
Coleopteran-active strains, such as B.t. M-7, B.t. PS86B1, and B.t. PS43F, can be used to control foliar-feeding beetles. The Colorado potato beetle (Leptinotarsa decemlineata), for example, is susceptible to the .delta.-endotoxin of B.t. M-7 and larvae are killed upon ingesting a sufficient dose of spore/crystal preparation on treated foliage.
Hofte and Whiteley (Hofte, H., H. R. Whiteley [1989] Microbiological Reviews 52(2):242-255) classified B.t. crystal protein genes into four major classes. The classes were CryI (Lepidoptera-specific), CryII (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported (Feitelson, J. S., J. Payne, L. Kim [1992] Bio/Technology 10:271-275). CryV was proposed to designate a class of toxin genes that are nematode-specific. Other classes of B.t. genes have now been identified.
The 1989 nomenclature and classification scheme of Hofte and Whiteley for crystal proteins was based on both the deduced amino acid sequence and the host range of the toxin. That system was adapted to cover 14 different types of toxin genes which were divided into five major classes. As more toxin genes were discovered, that system started to become unworkable, as genes with similar sequences were found to have significantly different insecticidal specificities. A revised nomenclature scheme has been proposed which is based solely on amino acid identity (Crickmore et al. [1996] Society for Invertebrate Pathology, 29th Annual Meeting, 3rd International Colloquium on Bacillus thuringiensis, University of Cordoba, Cordoba, Spain, September 1-6, abstract). The mnemonic "cry" has been retained for all of the toxin genes except cytA and cytB, which remain a separate class. Roman numerals have been exchanged for Arabic numerals in the primary rank, and the parentheses in the tertiary rank have been removed. Many of the original names have been retained, with the noted exceptions, although a number have been reclassified.
B.t. isolate PS86A1 is disclosed in the following, U.S. Pat. Nos. 4,849,217 (activity against alfalfa weevil); 5,208,017 (activity against corn rootworm); 5,286,485 (activity against lepidopterans); and 5,427,786 (activity against Phyllotreta genera). A gene from PS86A1 was cloned into B.t. MR506, which is disclosed in U.S. Pat. No. 5,670,365 (activity against nematodes) and PCT international patent application publication no. WO93/04587 (activity against lepidopterans). The sequences of a gene and a Cry6A (CryVIA) toxin from PS86A1 are disclosed in the following U.S. Pat. Nos. 5,186,934 (activity against Hypera genera); 5,273,746 (lice); 5,262,158 and 5,424,410 (activity against mites); as well as in PCT international patent application publication no. WO94/23036 (activity against wireworms). U.S. Pat. Nos. 5,262,159 and 5,468,636, disclose PS86A1, the sequence of a gene and toxin therefrom, and a generic formula for toxins having activity against aphids.
B.t. isolate PS52A1 is disclosed by the following U.S. Pat. Nos. as being active against nematodes: 4,861,595; 4,948,734, 5,093,120, 5,262,399, 5,236,843, 5,322,932; and 5,670,365. PS52A1 is also disclosed in 4,849,217, supra, and PCT international patent application publication no. WO95/02694 (activity against Calliphoridae). The sequences of a gene and a nematode-active toxin from PS52A1 are disclosed in U.S. Pat. No. 5,439,881 and European patent application publication no. EP 0462721. PS52A1, the sequence of a gene and nematode-active toxin therefrom, and a generic formula for CryVIA toxins are disclosed in PCT international patent application publication no. WO 92/19739.
As a result of extensive research, other patents have issued for new B.t. isolates and new uses of B.t. isolates. However, the discovery of new B.t. isolates, toxins, and genes, and new uses of known B.t. isolates remains an empirical, unpredictable art.
Although B.t. strains PS86A1 and PS52A1, and a gene and toxin therefrom, were known to have certain pesticidal activity, additional genes encoding active toxins from these isolates were not previously known in the art.